The present invention relates to the treatment of the cardiovascular system and its diseases through effects on anatomy, conduit function, and permeability, and more particularly to a method of treating cardiovascular disease by stimulating vascular cell proliferation using a growth factor thereby stimulating endothelial cell growth and vascular permeability.
Cardiovascular diseases are generally characterized by an impaired supply of blood to the heart or other target organs. Myocardial infarction (MI), commonly referred to as heart attacks, are a leading cause of mortality as 30% are fatal in the in the first months following the heart attack. Heart attacks result from narrowed or blocked coronary arteries in the heart which starves the heart of needed nutrients and oxygen. When the supply of blood to the heart is compromised, cells respond by generating compounds that induce the growth of new blood vessels so as to increase the supply of blood to the heart. These new blood vessels are called collateral blood vessels. The process by which new blood vessels are induced to grow out of the existing vasculature is termed angiogenesis, and the substances that are produced by cells to induce angiogenesis are the angiogenic factors.
Unfortunately, the body""s natural angiogenic response is limited and often inadequate. For this reason, the discovery of angiogenic growth factors has lead to the emergence of an alternative therapeutic strategy which seeks to supplement the natural angiogenic response by supplying exogenous angiogenic substances.
Attempts have been made to stimulate angiogenesis by administering various growth factors. U.S. Pat. No. 5,318,957 to Cid et al. discloses a method of stimulating angiogenesis by administering haptoglobins (glyco-protein with two polypeptide chains linked by disulfide bonds). Intracoronary injection of a recombinant vector expressing human fibroblast growth factor-5 (FGF-5) (i.e., in vivo gene transfer) in an animal model resulted in successful amelioration of abnormalities in myocardial blood flow and function. (Giordano, F. J.,et. al. Nature Med 2, 534-539, 1996). Recombinant adenoviruses have also been used to express angiogenic growth factors in-vivo. These included acidic fibroblast growth factor (Muhlhauser, J., et. al. Hum. Gene Ther. 6, 1457-1465, 1995), and one of the VEGF forms, VEGF165 (Muhlhauser, J., et. al. Circ. Res. 77, 1077-1086, 1995).
One of the responses of heart muscle cells to impaired blood supply involves activation of the gene encoding Vascular Endothelial Growth Factor (xe2x80x9cVEGFxe2x80x9d) (Banai, S., et. al. Cardiovasc. Res. 28:1176-1179, 1994). VEGFs are a family of angiogenic factors that induce the growth of new collateral blood vessels. The VEGF family of growth factors are specific angiogenic growth factors that target endothelial (blood vessel-lining) cells almost exclusively. (Reviewed in Ferrara et al., Endocr. Rev. 13:18-32 (1992); Dvorak et al., Am. J. Pathol. 146:1029-39 (1995); Thomas, J. Biol. Chem. 271:603-06 (1996)). Expression of the VEGF gene is linked in space and time to events of physiological angiogenesis, and deletion of the VEGF gene by way of targeted gene disruption in mice leads to embryonic death because the blood vessels do not develop. It is therefore the only known angiogenic growth factor that appears to function as a specific physiological regulator of angiogenesis.
In vivo, VEGFs induce angiogenesis (Leung et al., Science 246:1306-09, 1989) and increase vascular permeability (Senger et al., Science 219:983-85, 1983). VEGFs are now known as important physiological regulators of capillary blood vessel formation. They are involved in the normal formation of new capillaries during organ growth, including fetal growth (Peters et al., Proc. Natl. Acad. Sci. USA 90:8915-19, 1993), tissue repair (Brown et al., J. Exp. Med. 176:1375-79, 1992), the menstrual cycle, and pregnancy (Jackson et al., Placenta 15:341-53, 1994; Cullinan and Koos, Endocrinology 133:829-37, 1993; Kamat et al., Am. J. Pathol. 146:157-65, 1995). During fetal development, VEGFs appear to play an essential role in the de novo formation of blood vessels from blood islands (Risau and Flamme, Ann. Rev. Cell. Dev. Biol. 11:73-92, 1995), as evidenced by abnormal blood vessel development and lethality in embryos lacking a single VEGF allele (Carmeliet et al., Nature 380:435-38, 1996). Moreover, VEGFs are implicated in the pathological blood vessel growth characteristic of many diseases, including solid tumors (Potgens et al., Biol. Chem. Hoppe-Seyler 376:57-70, 1995), retinopathies (Miller et al., Am. J. Pathol. 145:574-84, 1994; Aiello et al., N. Engl. J. Med. 331:1480-87, 1994; Adamis et al., Am. J. Ophthalmol. 118:445-50, 1994), psoriasis (Detmar et al., J. Exp. Med. 180:1141-46, 1994), and rheumatoid arthritis (Fava et al., J. Exp. Med. 180:341-46, 1994).
Using the rabbit chronic limb ischemia model, it has been shown that repeated intramuscular injection or a single intra-arterial bolus of VEGF can augment collateral blood vessel formation as evidenced by blood flow measurement in the ischemic hindlimb (Pu, et al., Circulation 88:208-15, 1993; Bauters et al., Am. J. Physiol. 267:H1263-71, 1994; Takeshita et al., Circulation 90 [part 2], II-228-34, 1994; Bauters et al., J. Vasc. Surg. 21:314-25, 1995; Bauters et al., Circulation 91:2802-09, 1995; Takeshita et al., J. Clin. Invest. 93:662-70, 1994). In this model, VEGF has also been shown to act synergistically with basic FGF to ameliorate ischemia (Asahara et al., Circulation 92:[suppl 2], II-365-71, 1995). VEGF was also reported to accelerate the repair of balloon-injured rat carotid artery endothelium while at the same time inhibiting pathological thickening of the underlying smooth muscle layers, thereby maintaining lumen diameter and blood flow (Asahara et al., Circulation 91:2793-2801, 1995). VEGF has also been shown to induce EDRF (Endothelin-Derived Relaxin Factor (nitric oxide))-dependent relaxation in canine coronary arteries, thus potentially contributing to increased blood flow to ischemic areas via a secondary mechanism not related to angiogenesis (Ku et al., Am. J. Physiol. 265:H586-H592, 1993).
Activation of the gene encoding VEGF results in the production of several different VEGF variants, or isoforms, produced by alternative splicing wherein the same chromosomal DNA yields different mRNA transcripts containing different exons thereby producing different proteins. Such variants have been disclosed, for example, in U.S. Pat. No. 5,194,596 to Tischer et al. which identifies human vascular endothelial cell growth factors having peptide sequence lengths of 121, and 165 amino acids (i.e., VEGF121 and VEGF165). Additionally, VEGF189 and VEGF206 have also been characterized and reported (Neufeld, G., et. al. Cancer Metastasis Rev. 15:153-158, 1996).
As depicted in FIG. 1, the domain encoded by exons 1-5 contains information required for the recognition of the known VEGF receptors KDR/flk-1 and flt-1 (Keyt, B. A., et. al. J. Biol Chem 271:5638-5646, 1996), and is present in all known VEGF isoforms. The amino-acids encoded by exon 8 are also present in all known isoforms. The isoforms may be distinguished however by the presence or absence of the peptides encoded by exons 6 and 7 of the VEGF gene, and the presence or absence of the peptides encoded by these exons results in structural differences which are translated into functional differences between the VEGF forms (reviewed in: Neufeld, G., et. al. Cancer Metastasis Rev. 15, 153-158, 1996).
Exon 6 can terminate after 72 bp at a donor splice site wherein it contributes 24 amino acids to VEGF forms that contain it such as VEGF189. This exon 6 form is referred to as exon 6a. However, the VEGF RNA can be spliced at the 3xe2x80x2 end of exon 6 using an alternative splice site located 51 bp downstream to the first resulting in a larger exon 6 product containing 41 amino-acids. The additional 17 amino-acids added to the exon 6 product as a result of this alternative splicing are referred to herein as exon 6b. VEGF206 contains the elongated exon 6 composed of 6a and 6b, but this VEGF form is much rarer than VEGF189. (Tischer, E., et al., J. Biol. Chem. 266, 11947-11954; Houck, K. A., et al., Mol. Endocrinol., 12, 1806-1814, 1991).
A putative fifth form of VEGF, VEGF145, has been noted in the human endometrium, using PCR. The authors state that the sequence of the cDNA of the VEGF145 splice variant indicated that it contained exons 1-5, 6 and 8. However, it is uncertain whether the authors found that the splice variant contained exons 6a and 6b as in VEGF206, exon 6a as in VEGF189, or exon 6b. The authors state that since the splice variant retains exon 6 it is probable that it will be retained by the cell as are the other members of the family that contain this exon. (Charnock-Jones et al., Biology of Reproduction 48, 1120-1128 (1993). See also, Bacic M, et al. Growth Factors 12, 11-15, 1995). The biologic activity of this form has not heretofore been established. (Cheung, C. Y., et al., Am J. Obstet Gynecol., 173, 753-759, 1995); Anthony, F. W. et al., Placenta, 15, 557-561, 1994). The various isoforms, and the exons that encode the isoforms, are depicted in FIG. 1.
The four known forms of VEGF arise from alternative splicing of up to eight exons of the VEGF gene (VEGF121, exons 1-5,8; VEGF165, exons 1-5,7,8; VEGF189, exons 1-5, 6a, 7, 8; VEGF206, exons 1-5, 6a, 6b, 7, 8 (exon 6a and 6b refer to 2 alternatively spliced forms of the same exon)) (Houck et al., Mol. Endocr., 5:1806-14 (1991)). All VEGF genes encode signal peptides that direct the protein into the secretory pathway. For example, VEGF165 cDNA encodes a 191-residue amino acid sequence consisting of a 26-residue secretory signal peptide sequence, which is cleaved upon secretion of the protein from cells, and the 165-residue mature protein subunit. However, only VEGF121 and VEGF165 are found to be readily secreted by cultured cells whereas VEGF189 and VEGF206 remain associated with the producing cells. These VEGF forms possess an additional highly basic sequence encoded by exon 6 corresponding to residues 115-139 in VEGF189 and residues 115-156 in VEGF206. These additions confer a high affinity to heparin and an ability to associate with the extracellular matrix (matrix-targeting sequence) (Houck, K. A. et al., J. Biol. Chem. 267:26031-37 (1992) and Thomas, J. Biol. Chem. 271:603-06 (1996)). The mitogenic activities of VEGF121 and VEGF165 are similar according to the results of several groups (Neufeld, G., et al., Cancer Metastasis Rev. 15:153-158 (1996) although one research group has shown evidence indicating that VEGF121 is significantly less active (Keyt, B. A., et al., J. Biol. Chem. 271:7788-7795 (1996). It is unclear whether the two longer VEGF forms, VEGF189 and VEGF206, are as active or less active than the two shorter forms since it has not been possible to obtain them in pure form suitable for quantitative measurements. This failure is due in part to their strong association with producing cells and extracellular matrices which is impaired by the presence of exon-6 derived sequences apparently acting in synergism with exon-7 derived sequences groups (Neufeld, G., et al., Cancer Metastasis Rev. 15:153-158 (1996).
As described in more detail herein, each of the VEGF splice variants that have heretofore been characterized have one or more of the following disadvantages with respect to stimulating angiogenesis of endothelial cells in the treatment of cardiovascular diseases: (i) failure to bind to the extracellular matrix (ECM) resulting in faster clearance and a shorter period of activity, (ii) failure to secrete into the medium (i.e. remaining cell-associated) so as to avoid reaching and acting on the endothelial cells, and (iii) susceptibility to oxidative damage thereby resulting in shorter half-life.
Accordingly, there is a need for a new form of VEGF that avoids the aforementioned disadvantages and that can be usefully applied in stimulating angiogenesis in cardiovascular disease patients would be most desirable.
The present invention relates to a novel VEGF protein product, and a nucleic acid encoding the novel protein product comprising exons 1-6a and 8 of the VEGF gene, (hereinafter xe2x80x9cVEGF145xe2x80x9d) and the use thereof in treating the cardiovascular system and its diseases through effects on anatomy, conduit function, and permeability. VEGF145 has been found to be an active mitogen for vascular endothelial cells and to function as an angiogenic factor in-vivo. VEGF145 was favorably compared with previously characterized VEGF species with respect to cellular distribution, susceptibility to oxidative damage, and extra-cellular matrix (ECM) binding ability.
Previous research relating to the binding affinities of the various VEGF isoforms found that VEGF165, which lacks exon 6, binds relatively weakly to heparin and also binds very weakly to the extracellular matrix, (Park, J. E., et al., Mol. Biol. Cell 4:1317-1326 (1993). VEGF145, which binds as weakly as VEGF165 to heparin, binds much better than VEGF165 to the extracellular matrix. However, unlike VEGF189, VEGF145 is secreted from producer cells and binds efficiently to the ECM. This combination of properties render VEGF145 the only known VEGF variant that is secreted from producing cells retaining at the same time extracellular matrix binding properties. Hence, it will likely diffuse towards the target blood vessels, while some of the produced VEGF145 will be retained by extracellular matrix components along the path of diffusion. This ECM bound pool will dissociate slowly allowing a longer period of activity. Furthermore, the biological activity of VEGF145 is protected against oxidative damage unlike VEGF forms such as VEGF121 thereby giving it a longer half-life.
In sum, VEGF145 clearly possesses a unique combination of biological properties that distinguish it from the other VEGF forms. This unique combination of properties of VEGF145 renders it a preferred therapeutic agent for the treatment of the cardiovascular system and its diseases as well as other diseases characterized by vascular cell proliferation. In particular, the cDNA may be employed in gene therapy for treating the cardiovascular system and its diseases.
Endothelial cell proliferation, such as that which occurs in angiogenesis, is also useful in preventing restenosis following balloon angioplasty. The balloon angioplasty procedure often injuries the endothelial cells lining the inner walls of blood vessels. Smooth muscle cells often infiltrate into the opened blood vessels causing a secondary obstruction in a process known as restenosis. The proliferation of the endothelial cells located at the periphery of the balloon-induced damaged area in order to cover the luminal surface of the vessel with a new monolayer of endothelial cells would potentially restore the original structure of the blood vessel.
Thus, the present invention provides a method of treating cardiovascular disease in a mammal comprising the step of transfecting cells of said mammal with a polynucleotide which encodes VEGF145. In preferred aspects, the polynucleotide is cloned into a vector. In further preferred aspects, the vector is an adenovirus vector. The adenovirus vector is preferably delivered to the mammal by injection; preferably, about 1010 to about 1014 adenovirus vector particles are delivered in the injection. More preferably, about 1011 to about 1013 adenovirus vector particles are delivered in the injection. Most preferably, about 1012 adenovirus vector particles are delivered in the injection.
In further preferred aspects, the polynucleotide which encodes VEGF145 is delivered to the heart of a mammal. The delivery of the polynucleotide is preferably by intracoronary injection into one or both arteries, preferably according to the methods set forth in PCT/US96/02631, published Sep. 6, 1996 as WO96/26742, hereby incorporated by reference herein. Preferably, the intracoronary injection is conducted about 1 cm into the lumens of the left and right coronary arteries.
In other preferred aspects of the invention, the cells of the mammal are transfected in vivo. In other preferred aspects, the cells are transfected ex vivo.
In yet other preferred aspects of the invention, the polynucleotide may be introduced into the mammal through a catheter.
In one embodiment of the invention, the polynucleotide which encodes VEGF145 comprises a base sequence as defined in the Sequence Listing by SEQ ID No. 1. In preferred embodiments, the polynucleotide sequence encoding VEGF145 is present in an expression vector. Thus, in a preferred aspect of the invention, the invention provides an expression vector comprising a polynucleotide sequence encoding VEGF145 species, said species being selected from the group consisting of:
(a) VEFG145;
(b) a biologically active fragment of VEGF145; and
(c) a biologically active derivative of VEGF145, wherein an amino acid residue has been inserted, substituted or deleted in or from the amino acid sequence of the VEGF145 or its fragment. In preferred aspects, the polynucleotide encodes VEGF145. In more preferred aspects, the polynucleotide comprises a base sequence as defined in the Sequence Listing by SEQ ID No. 1.
In a preferred embodiment of the invention, the polynucleotide encoding VEGF145 is present in an adenovirus expression vector, thus, in preferred aspects, the polynucleotide is flanked by adenovirus sequences. In yet other preferred aspects, the polynucleotide sequence is operably linked at its 5xe2x80x2 end to a promoter sequence that is active in vascular endothelial cells. In preferred expression vectors, the expression vector further comprises a partial adenoviral sequence from which the EIA/EIB genes have been deleted.
Also provided in the present invention are kits for intracoronary injection of a recombinant vector expressing VEGF145 comprising:
a polynucleotide encoding VEGF145 cloned into a vector suitable for expression of said polynucleotide in vivo,
a suitable container for said vector, and
instructions for injecting said vector into a patient. In more preferred aspects, the polynucleotide is cloned into an adenovirus expression vector.
In other preferred embodiments of the invention, the methods, compositions, and vectors of the present invention may be used to treat cardiovascular disease in a mammal comprising the step of administering to said mammal VEGF145 in a therapeutically effective amount to stimulate angiogenesis. In other preferred embodiments, the methods, compositions, and vectors of the present invention may be used to treat vascular disease in a mammal comprising the step of administering to said mammal VEGF145 in a therapeutically effective amount to stimulate vascular cell proliferation. In yet other preferred embodiments of the present invention, the methods, compositions, and vectors of the invention may be used to enhance endothelialization of diseased vessels comprising the step of administering to a mammal a therapeutically effective amount of VEGF145. Preferably, endothelialization comprises reendothelialization after angioplasty, to reduce or prevent restenosis. Those of skill in the art will recognize that patients treated according to the methods of the present invention may be treated with or without a stent.
In yet other preferred embodiments of the present invention, the methods, compositions, and vectors of the invention may be used to enhance drug permeation by tumors comprising administering to a patient a nucleic acid molecule coding for VEGF145. The VEGF145 may be delivered directly to a tumor cell, or it may be delivered into the vascular system, preferably at a site located close to the site of the tumor. Thus, delivery of VEGF145 in conjunction with chemotherapy to remove or reduce the size of a tumor, will help to enhance the effectiveness of the chemotherapy by increasing drug uptake by the tumor. The VEGF145 delivered in this method may either be through direct delivery of the polypeptide or protein, or through gene therapy.
In another embodiment of the invention is provided a therapeutic composition comprising a pharmaceutically acceptable carrier and VEGF145 in a therapeutically effective amount to stimulate vascular cell proliferation.
In other preferred embodiments of the invention is provided a filtered injectable adenovirus vector preparation, comprising: a recombinant adenoviral vector, said vector containing no wild-type virus and comprising:
a partial adenoviral sequence from which the E1A/E1B genes have been deleted, and
a transgene coding for a VEGF145, driven by a promoter flanked by the partial adenoviral sequence; and
a pharmaceutically acceptable carrier.
In other preferred aspects, the invention provides a recombinant plasmid comprising a polynucleotide which codes for VEGF145. In yet other preferred aspects, the invention provides a transformed microorganism transformed with the recombinant plasmid.
With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the invention, the figures, and the appended claims.